Project Summary Most bacteria maintain a cell wall, an essential, mesh-like structure mainly comprising the polysaccharide peptidoglycan (PG). Some of our most powerful antibiotics, the beta lactams (penicillins, carbapenems and cephalosporins) target enzymes required for cell wall synthesis and derive their efficacy from their ability to not only inhibit cell wall biogenesis, but also to actively cause its disruption. Cell wall disruption after exposure to beta lactams is mediated by ?autolysins?, a group of enzymes (amidases, lytic transglycosylases and endopeptidase) with the capacity to cut a variety of chemical bonds within the PG mesh. Under normal growth conditions, autolysins engage in important cell wall remodeling functions, such as PG mesh expansion during cell elongation; how these functions are regulated to ensure proper PG maintenance is poorly understood. We have shown that in the diarrheal pathogen Vibrio cholerae, the endopeptidases (EPs) ShyA and ShyC are required for cell elongation during normal growth (the physiological functions of another paralogue, ShyB, are unknown), but are also key factors mediating cell wall breakdown after exposure to beta lactam antibiotics. How ShyA and ShyC are regulated to ensure proper cell wall maintenance in the absence of antibiotics is unknown. Here, we propose experiments to build a thorough understanding of mechanisms of endopeptidase regulation in V. cholerae on multiple levels. Since M23 EPs are well-conserved throughout Bacteria, our experiments will likely yield insights with broad relevance to other pathogens. In Aim 1, we will dissect the functional relationship between PG synthesis and degradation processes. We will also test the hypothesis that ShyA and ShyC's direct interaction with cell wall synthesis complexes regulates their activity. In Aim 2 we will precisely map structure-function relationships in EPs and discover additional regulators of their activity. In Aim 3 we will determine the mechanism of EP regulation by metal homeostasis. Taken together, these experiments will provide us with an extensive framework of how an important human pathogen maintains the balance between cell wall synthesis and remodeling, with the goal of discovering new potential targets for antibiotics that modulate autolysin activity.